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Coexpression of Human Somatostatin Receptor-2 (SSTR2) and SSTR3 Modulates Antiproliferative Signaling and Apoptosis Sajad a War and Ujendra Kumar*

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Coexpression of Human Somatostatin Receptor-2 (SSTR2) and SSTR3 Modulates Antiproliferative Signaling and Apoptosis Sajad a War and Ujendra Kumar* War and Kumar Journal of Molecular Signaling 2012, 7:5 http://www.thrombosisjournal.com/7/1/5 RESEARCH ARTICLE Open Access Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis Sajad A War and Ujendra Kumar* Abstract Background: Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3. Results: Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect. Conclusion: Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology. Keywords: Apoptosis, Photobleaching-Fluorescence resonance energy transfer (Pb-FRET), G protein-coupled receptor (GPCR), Heterodimerization, Somatostatin, Somatostatin receptors Background subtypes display receptor-specific homo- and/or heterodi- G protein-coupled receptors (GPCRs) assemble as oligo- merization within the family and other GPCRs with mers with distinct pharmacological, biochemical and unique signaling characteristics [3,6-9]. This notion is fur- physiological properties [1-3]. The concept of oligomeri- ther supported by our previous studies demonstrating zation with efficacious changes in downstream signaling enhanced signaling properties in the heteromeric complex pathways have broadened the therapeutic potential of drugs of human SSTR2 or SSTR5 with dopamine receptor-2 targeting GPCRs. Somatostatin (SST) is a pleiotropic inhibi- [10,11]. Interestingly, the heterodimer of SSTR2/SSTR3 of tory peptide and regulates endocrine and exocrine secre- rat origin has been reported to abrogate SSTR3 functions tions, neurotransmission and cell proliferation through [12]. However, rat SSTRs show distinct response to agonist five different receptor subtypes coupled to Gi proteins in comparison to human SSTRs, [9,12-14]. namely somatostatin receptors (SSTR1-5) [4,5]. SSTR All SSTR subtypes upon activation couple to Gi pro- teins and inhibit adenylyl cyclase (AC) in a pertussis * Correspondence: [email protected] toxin (PTX)-sensitive manner [4]. Importantly, in cells Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BCV6T 1Z3, Canada coexpressing human SSTR2 and SSTR5, cyclic adenosine © 2012 War and Kumar; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 2 of 15 http://www.thrombosisjournal.com/7/1/5 monophosphate (cAMP) inhibition was enhanced upon ac- subtypes [4]. Although, rat SSTR2/SSTR3 heterodimeri- tivation of SSTR2 [3]. Conversely, cAMP levels remained zation abolished the functions of SSTR3 [12], the same unchanged in response to SSTR3-specific agonist in cells may not be speculated for human SSTR2 and SSTR3 as cotransfected with rat SSTR2 and SSTR3, whereas the ac- receptors of different origin have diverse signaling prop- tivation of SSTR2 caused significant inhibition of cAMP erties and need to be elucidated in detail. In this study, [12]. We and others have shown the role of SSTRs in regu- using morphological, biochemical and biophysical tech- lating intracellular signaling molecules including mitogen- niques, we provide first evidence for human SSTR2/ activated protein kinases (MAPKs) which are implicated SSTR3 heterodimerization in human embryonic kidney in cell proliferation, differentiation, migration and apop- (HEK-293) cells and its implications in signaling and tosis [3,9,15-20]. The antiproliferative response of SSTRs antiproliferation. is associated with the phosphorylation of selective down- stream cascades including extracellular signal-regulated Materials and methods kinases (ERKs) depending upon the receptor subtype, cell Materials environment and extracellular factors [3,4,8,9,21-23]. ERK Non-peptide agonists for SSTR2 (L-779976) and SSTR3 is activated by SSTR1 and SSTR4, whereas inhibited (L-796778) were kindly provided by Dr. S.P. Rohrer from upon activation of SSTR5 [8,24-26]. On the other hand, Merck & Co [39]. SST was purchased from Bachem Inc., SSTR2 and SSTR3 exhibit dual effect on ERK phosphoryl- Torrance, CA, USA. HEK-293 cells were obtained from ation in a cell-specific manner [4,9,12,22,25,27]. In the oli- ATCC, Manassas, VA, USA. Rabbit polyclonal antibodies gomeric complex of human SSTR2/SSTR5 or rat SSTR2/ against SSTR2 were generated and characterized as SSTR3, ERK1/2 phosphorylation has been attributed described previously [40]. Mouse monoclonal antibodies to SSTR2 activation [3,12]. Alterations in stress-related p38 against p21 and PARP-1 were purchased from BD- MAPK have been frequently observed in human tumors Biosciences, Mississauga, ON, Canada. Rabbit polyclonal and various other cell lines of tumor origin [28,29]. p38 sig- antibodies for total and phospho-ERK1/2 and p38 naling has diverse biological consequences including pro-/ were obtained from Cell Signaling Technology, Danvers, anti-apoptotic effects in a cell-dependent manner [27-29]. MA, USA. Fluorescein and rhodamine or peroxidase con- Importantly, the antiproliferative response mediated by jugated goat anti-mouse and goat anti-rabbit second- SSTR2 but not SSTR3 has been associated with the ary antibodies were purchased from Jackson Immuno activation of p38 MAPK [27]. Inhibition of cell prolifera- Research Laboratories, West Grove, PA, USA). Mouse tion by SSTR subtypes engages multiple converging monoclonal antibodies for p27Kip1 were obtained from mechanisms, however SSTR2 and SSTR3 are specific- Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. ally linked to cell cycle arrest and apoptosis, respectively Mouse monoclonal antibodies against hemagglutinin [3,4,9,30-35]. (HA) and β-Tubulin were purchased from Sigma-Aldrich Agonist-induced internalization of SSTRs is time, Inc., St. Louis, MO, USA. cAMP kit was obtained from temperature, and receptor-specific; however heterodi- BioVision Inc., Milpitas, CA, USA. TUNEL kit was pur- merization plays a determinant role on receptor traffick- chased from La Roche Applied Science, Mannheim, ing [36,37]. Importantly, agonist-mediated internalization Germany. ECL Western blotting detection kit and nitrocel- of SSTRs varies significantly between receptors of rat lulose Hy-Bond ECL membrane were purchased from and human origin [9,12,13,36]. SSTR1 of rat origin inter- GE Healthcare, Piscataway, NJ, USA. Protein A/G- nalized in response to agonist, whereas human SSTR1 Agarose beads were obtained from Calbiochem, EMD rather up-regulated at the cell surface [36,38]. The con- Biosciences, Darmstadt, Germany. Dulbecco’smodified stitutive homodimer of human SSTR2 dissociated into eagle medium (DMEM), trypsin-EDTA, phosphate buf- monomers at the plasma membrane prior to agonist- fered saline (PBS) were purchased from Invitrogen, stimulated internalization, whereas rat SSTR2 interna- Burlington, ON, Canada. Reagents for electrophoresis lized as homodimer [6,12]. On the other hand, human were obtained from Bio-Rad Laboratories, Mississauga, and rat SSTR3 internalized as homodimers upon agonist ON, Canada. Other reagents were of AR grade and treatment [9,12]. Strikingly, rat SSTR3 internalized and were purchased from various sources. subsequently recycled to the cell surface in response to agonist, whereas human SSTR3 was targeted to degrad- Human SSTR2 and SSTR3 constructs and transfection ation [13,14,36]. More importantly, C-tail of human Human SSTR2 construct was prepared using pCDNA3.1/ SSTR3 was not essential for receptor trafficking, con- Hyg vector (hygromycin resistance). Constructs expressing versely, mutations in C-terminal of rat SSTR3 abrogated human SSTR3 and N-terminal HA-tagged human SSTR3 the agonist-mediated internalization [9,13]. (HA-SSTR3) were prepared using pCDNA3.1/Neo vector SSTR3 shares the least amino acid homology between (neomycin resistance),
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